Water‐in‐Water Droplets Selectively Uptake Self‐Assembled DNA Nano/Microstructures: a Versatile Method for Purification in DNA Nanotechnology

Abstract DNA is an excellent material for constructing self‐assembled nano/microstructures. Owing to the widespread use of DNA as a building block in laboratories and industry, it is desirable to increase the efficiency of all steps involved in producing self‐assembled DNA structures. One of the bottlenecks is the purification required to separate the excess components from the target structures. This paper describes a purification method based on the fractionation by water‐in‐water (W/W) droplets composed of phase‐separated dextran‐rich droplets in a polyethylene glycol (PEG)‐rich continuous phase. The dextran‐rich droplets facilitate the selective uptake of self‐assembled DNA nano/microstructures and allow the separation of the target structure. This study investigates the ability to purify DNA origami, DNA hydrogels, and DNA microtubes. The W/W‐droplet fractionation allows the purification of structures of a broad size spectrum without changes to the protocol. By quantifying the activity of deoxyribozyme‐modified DNA origami after W/W‐droplet purification, this study demonstrates that this method sufficiently preserves the accessibility to the surface of a functional DNA nanostructure. It is considered that the W/W‐droplet fractionation could become one of the standard methods for the purification of self‐assembled DNA nano/microstructures for biomedical and nanotechnology applications owing to its low cost and simplicity.


HEXAGONAL DNA ORIGAMI
The DNA origami with the shape of a hexagonal nanoplate was used to initially test the viability of W/W-droplet fractionation to purify DNA structures. The DNA origami was composed of six interconnected trapezoidal structures named P1-P6 (Fig. S1). Each trapezoidal portion contains staples in positions L1 and L2. We produced a version of the DNA origamis with staples in positions L1 and L2 extended with an 8-17 deoxirybozyme (DNAzyme), totaling 12 DNAzymes per DNA origamis. The table S1 shows the list of staples used for producing the non-enzymatic hexagonal DNA origami and their length, relative start/end in the scaffold and indicating whether they belong to the L1 or L2 position. . Table   S2 indicates the staples used for producing the enzymatic DNA origami, showing only the staples that substitute those in positions L1 and L2. Figure S1. Structure of hexagonal DNA origami, adapted from Ishikawa et al. [1] Supplementary Table S1. The sequence of oligonucleotides used in the DNA origami with a hexagonal shape. The columns indicate the staple number, sequence, length, relative start and end, and indicate if the staple belongs to the L1 or L2 position indicated in Fig. S1 Table S1, which occupy the positions L1 and L2.  Table S3. In the next section, we describe the preparation protocol of each structure. The preparation of the DNA origami using the reduced scaffold followed the protocol by Said et al. [4] In this protocol, a linear section of the circular M13mp18 ssDNA (Tilibit

TRIANGLE, 24HB DNA ORIGAMI
The staples of the triangular DNA origami were obtained from the original report by Rothemund [3] and were prepared under the same conditions as the hexagonal DNA origami using staples from Eurofins Genomics (salt-free grade). The 3D DNA structure 24HB was composed of 10 nM scaffold p7560 (Tilibit Nanosystems, lot M1-3-4), 100 nM of staples (OPC grade from Eurofins genomics) as designed by Kuzbyk et al. [5] , 1✕TAE buffer and 16 mM magnesium acetate. The annealing program consisted of heating the sample to 80°C for 10 min and cooling it from 80°C to 65°C at -1°C min -1 , 65°C to 45°C at -0.1°C min -1 and 45°C to 25°C at -0.05°C min -1 .

STICKY-END-LESS Y-MOTIF AND DNA HYDROGEL
The Y-motif structure is a kind of DNA motif composed of a set of 3 oligonucleotides.
We used Y-motifs that were both unable to form DNA hydrogel (sticky-end-less Y-motif) and those which were able to form a hydrogel, the difference being a palindromic sequence  Table S4. They were obtained from Eurofins Genomics (salt-free grade) and mixed with 1✕TAE buffer, 100 mM potassium chloride, 10 mM sodium chloride, 0.5 mM magnesium chloride, and 0.1 mM calcium chloride. The sample was heated to 95°C for 3 minutes, then cooled to 25°C at -1°C min -1 .

4HT AND 13HT MICROTUBES
The 4HT and 13HT microtubes were composed of bundles of nanotubes assembled using DNA tile technology. The bundles varied in number of nanotubes, which created microtubes with different diameters. Microtubes with bundles of 4 and 13 nanotubes, called 4HT and 13HT, respectively, were made following the protocol of Yin et al. [7] . A mixture of staples from Eurofins genomics (OPC grade) with the final concentration of 1 µM each and 1✕TAE buffer and 12 mM magnesium acetate was annealed by heating to 80°C for 10 min and cooling from 80°C to 65°C at -1°C min -1 , 65°C to 45°C at -0.1°C min -1 and 45°C to 25°C at -0.05°C min -1 . Table S4. Oligonucleotide sequences used for making sticky-end-less Ymotif and DNA hydrogel [2] .

DERIVATION OF PURIFICATION YIELD
During the purification protocol, the dextran-rich droplets were separated by centrifugation. The bottom phase is the denser dextran, and the top phase is the less dense PEG. The PEG phase is then exchanged, leaving the dextran solution and part of the PEG phase in the tube. The following model takes this process into account to calculate how much of a structure remains after each purification round. By applying number of rounds, it is possible to obtain the purification yield. µL of the PEG phase were exchanged, which means 15 µL of the bottom remained after each purification round. In the last round, 5 µL were recovered so the volume was the same as the initial sample. We called the volume that remained at the bottom at each round ( . , ) , * , … , , > V $%& . We can rewrite the concentration of the structure after the first purification round ( ( ) considering the ratio of the substance that was recovered: The above equation can be written in terms of [ ] by inserting the equations for [ ] !"# and [ ] $%& : Which can be rewritten as: On the n th purification round:

01(
By recursively applying this equation, the n th round can be rewritten: which gives the purification yield by W/W-droplet fractionation for a structure given the parameters ( !"# , $%& ), the experimental protocol ( ( , ) , … , , ) and the structure partition coefficient ( ).

THEORETICAL SEPARATION EFFICIENCY
We numerically investigated the dependence of size on the purification efficiency of the DNA nano/microstructures, the efficiency being defined by Equation 1. To this end, we have adopted des Coundres' theory [8] . Des Coudres' theory is a general explanation for the distribution of structures in aqueous two-phase systems. It assumes that the complex interactions between the molecules, structures, and the polymer phases can be summarized by the interfacial free energy, which is given by = γ, where F is the interfacial free energy, A is the structure/molecule surface area, and γ is the surface tension between the structure/molecule and the polymer solution. When the structure/molecule is dispersed in PEG, we use the notation !"# = !"# , and when the structure/molecule is in the dextran phase, $%& = $%& . The partition coefficient ( ) of a structure/molecule is given by [9] :

Equation S2
≪ 1 indicates that the structure/molecule is portioned primarily in the dextran phase; ≫ 1 that the structure/molecule is partitioned primarily in the PEG phase; and ~1 that the structure/molecule is present in similar concentrations in both phases. An estimate for ∆ can be made if we consider a model in which the polymers are ideal and non-interacting, the polymer solution is dilute, and the structures are much larger than the polymers. In this case, the surface tension between the molecule, structure, and the polymer solution can be approximated as the surface tension of a hard wall and a polymer solution, given by the formula [10] : where ρ is the numeric concentration of the polymer and A is the polymer radius. To determine the numeric density of the polymers ρ, we assumed that the concentration of PEG was approximately 100 g L -1 and that of dextran was 200 g L -1 , based on the binodal curve of similar dextran-PEG ATPS [11] , which is 9.6 × 10 )B molecules m -3 in the PEG phase and 6 × 10 )* molecules m -3 . We considered the size of PEG ( A = 2.2 nm) [12] and dextran ( A = 9 nm) (formula for molecular weight to size provided by Aimar et al. [13] ). Based on these values, γ !"# = 97.9 µN m -1 , γ $%& = 25.04 µN m -1 and ∆ = 72.8 µN m -1 .
By knowing ∆ , the structure/molecule surface area, and the conditions of the dextran-rich droplets, one can calculate and the purification yield. The equation S1 gives the purification yield.

QUANTIFICATION OF AGAROSE ELECTROPHORESIS
The agarose electrophoresis provided information on the electrophoretic mobility of molecules and structures, which is closely related to their size, and allows the quantification of the extent of purification and the structural integrity of the structures. The images of the agarose electrophoresis were analyzed with ImageJ [14,15]  In this study, we focused on purifying low volume, low concentration samples purification of DNA origami. Under these conditions, W/W-droplet purification demonstrated to be a facile and high-yielding method. We compared the same conditions to molecular weight cut-off and gel filtration. Although Amicon and gel filtration can be high yielding methods, without optimization, these methods provided yields below 50%, as shown in Fig.   S4.

DNA ORIGAMI WITH DNAZYME ACTIVITY
We fabricated two different kinds of DNA origami, one without catalytic activity and one with catalytic activity. The difference between these two designs is that in the catalytic DNA origami, there are 12 staples in positions L1 and L2 (see Fig. S1), which were extended with an 8-17 deoxyribozyme, which can cleave a DNA-RNA chimeric substrate. For most purification experiments, DNA origami without catalytic activity was used. The DNA origami with catalytic activity (DNAzyme origami) was used to test if the purification methods affected the functional DNA origamis due to residual polymers.
The catalytic DNAzyme origami was purified with molecular weight cut-off and W/W-droplet fractionation, while the non-catalytic DNA origami was purified with W/Wdroplet fractionation. We observed the structural integrity of the DNA origami by agarose electrophoresis, as shown in Fig. S5. Prior to purification, both the DNAzyme and the DNA origami display a sharp band; however, after purification, the DNAzyme origami displays a smeared band. This occurs both when purified by molecular weight cut-off filtration and W/W-droplet fractionation. This likely indicates that the extended staples can induce partial aggregation of the DNAzyme origami.
We tested the catalytic activity of the DNAzyme origami purified by molecular weight cut-off and W/W-droplet fractionation on a DNA-RNA chimera substrate. As a negative control, a dummy molecule composed only of DNA was used. Free DNAzymes acted as a positive control, and DNA origami without catalytic activity was used as an additional negative control. The result is displayed in Fig. S6A. This figure shows that the free DNAzyme, the DNAzyme origami purified by molecular weight cut-off, and DNAzyme origami purified by W/W-droplet fractionation retain their enzymatic function. Although it may be seen that the DNAzyme purified by W/W-droplet fractionation has a low activity due to the high fluorescence of the non-cleaved substrate, when the intensity profile of the lanes was quantified, we observed that the lanes where W/W-droplet purification was used had a fluorescence intensity one order of magnitude higher than lanes whose samples were not purified by W/W-droplet fractionation. That is, the W/W-droplet purification caused an increase in the fluorescence of the TAMRA dye, which makes it seem that the activity of DNAzyme origami purified by W/W-droplet fractionation is smaller than the DNAzyme origami purified by molecular weight cut-off. However, when the intensity of the lanes is normalized, and the relative intensity of bands is quantified, producing Fig. 3C, we observe that, in fact, free DNAzyme, DNAzyme origami purified by molecular weight cut-off, and DNAzyme origami purified by W/W-droplet fractionation have similar catalytic activity.
We confirmed that the W/W-droplet purification increases the fluorescence of the TAMRA dye by adding polymers dextran and PEG to the free DNAzyme and DNAzyme origami purified by molecular weight cut-off, as shown in Fig. S6B. In this figure, we can observe that the addition of polymers increases the fluorescence of the dye in more than one order of magnitude. When the intensity of the bands is normalized, we can confirm that the presence of polymers, nevertheless, does not affect the catalytic activity of the DNAzyme or the DNAzyme origami.